A soluble isoform of the rhesus monkey nonclassical MHC class I molecule Mamu-AG is expressed in the placenta and the testis

The nonclassical MHC class I locus HLA-G is expressed primarily in the placenta, although other sites of expression have been noted in normal and pathological situations. In addition, soluble HLA-G isoforms have been detected in the serum of pregnant and nonpregnant women as well as men. The rhesus monkey placenta expresses a novel nonclassical MHC class I molecule Mamu-AG, which has features remarkably similar to those of HLA-G. We determined that the rhesus placenta expresses Mamu-AG mRNA (Mamu-AG5), retaining intron 4 as previously noted in HLA-G5. Immunostaining experiments with Ab 16G1 against the soluble HLA-G5 intron 4 peptide demonstrated that an immunoreactive protein(s) was present in the syncytiotrophoblasts of the chorionic villi of the rhesus placenta, within villous cytotrophoblasts, and occasionally within cells of the villous stroma. The Mamu-AG5 mRNA was readily detected in rhesus testis (although not in ejaculated sperm). Whereas an Ab against membrane-bound Mamu-AG stained few cells, primarily in the interstitium of the testis, there was consistent immunostaining for Mamu-AG5 in cells within the seminiferous tubules, which was corroborated by localization of Mamu-AG mRNA by in situ hybridization. While primary spermatocytes were negative, Sertoli cells, spermatocytes, and spermatids were consistently positive for 16G1 immunostaining. The specific recognition of the soluble Mamu-AG isoform was confirmed by Western blotting of Mamu-AG5 expressed in heterologous cells. The results demonstrate that a soluble nonclassical MHC class I molecule is expressed in the rhesus monkey placenta and testis, and confirm and extend the unique homology between HLA-G and the rhesus nonclassical molecule Mamu-AG.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.     

Activation tagging using the En-I maize transposon system in Arabidopsis

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.     

Exogenous growth factors induce the production of ganglion cells at the retinal margin

Neural progenitors at the retinal margin of the post-hatch chicken normally produce amacrine and bipolar cells, but not photoreceptor or ganglion cells. The purpose of this study was to test whether exogenous growth factors influence the types of cells produced by progenitors at the retinal margin. We injected insulin, FGF2 or a combination of insulin and FGF2 into the vitreous chamber of post-hatch chickens. To assay for growth factor-induced changes at the retinal margin, we used in situ hybridization and immunocytochemistry on cryosections. One day after the final injection, we found that insulin alone stimulated the addition of cells to the retinal margin, but this was not further increased when FGF2 was applied with insulin. Insulin alone increased the number of cells in the progenitor zone that expressed neurofilament, and this was further increased when FGF2 was applied with insulin. These neurofilament-expressing cells in the progenitor zone included differentiating neurons that expressed Islet1 or Hu. Four days after the final dose of growth factor, we found that the production of ganglion cells was induced by co-injection of insulin and FGF2, but not by either insulin or FGF2 alone. We conclude that the types of cells produced by progenitors at the retinal margin can be altered by exogenous growth factors and that normally the microenvironment imposes limitations on the types of neurons produced.     

Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity

Asthma is caused by T-helper cell 2 (Th2)-driven immune responses, but the immunological mechanisms that protect against asthma development are poorly understood. T-cell tolerance, induced by respiratory exposure to allergen, can inhibit the development of airway hyperreactivity (AHR), a cardinal feature of asthma, and we show here that regulatory T (T(R)) cells can mediate this protective effect. Mature pulmonary dendritic cells in the bronchial lymph nodes of mice exposed to respiratory allergen induced the development of T(R) cells, in a process that required T-cell costimulation via the inducible costimulator (ICOS-ICOS-ligand pathway. The T(R) cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mice, T(R) cells blocked the development of AHR. Both the development and the inhibitory function of regulatory cells were dependent on the presence of IL-10 and on ICOS-ICOS-ligand interactions. These studies demonstrate that T(R) cells and the ICOS-ICOS-ligand signaling pathway are critically involved in respiratory tolerance and in downregulating pulmonary inflammation in asthma.     

A phylogenetic supertree of the bats (Mammalia: Chiroptera)

We present the first estimate of the phylogenetic relationships among all 916 extant and nine recently extinct species of bats Mammalia: Chiroptera), a group that accounts for almost one-quarter of extant mammalian diversity. This phylogeny was derived by combining 105 estimates of bat phylogenetic relationships published since 1970 using the supertree construction technique of Matrix Representation with Parsimony (MRP). Despite the explosive growth in the number of phylogenetic studies of bats since 1990, phylogenetic relationships in the order have been studied non-randomly. For example, over one-third of all bat systematic studies to date have locused on relationships within Phyllostomidae, whereas relationships within clades such as Kerivoulinae and Murinae have never been studied using cladistic methods. Resolution in the supertree similarly differs among clades: overall resolution is poor (46.4%, of a fully bifurcating solution) but reaches 100% in some groups (e.g. relationships within Mormoopidae). The supertree analysis does not support a recent proposal that Microchiroptera is paraphyletic with respect to Megachiroptera, as the majority of source topologies support microbat monophyly. Although it is not a substitute for comprehensive phylogenetic analyses of primary molecular and morphological data, the bat supertree provides a useful tool for future phylogenetic comparative and macroevolutionary studies. Additionally, it identifies clades that have been little studied, highlights groups within which relationships are controversial, and like all phylogenetic studies, provides preliminary hypotheses that can form starting points for future phylogenetic studies of bats.     

Mechanisms of metabotropic glutamate receptor desensitization: role in the patterning of effector enzyme activation

Metabotropic glutamate receptors (mGluRs) constitute an unique subclass of G protein-coupled receptors (GPCRs). These receptors are activated by the excitatory amino acid glutamate and play an essential role in regulating neural development and plasticity. In the present review, we overview the current understanding regarding the molecular mechanisms involved in the desensitization and endocytosis of Group 1 mGluRs as well as the relative contribution of desensitization to the spatial-temporal patterning of glutamate receptor signaling. Similar to what has been reported previously for prototypic GPCRs, mGluRs desensitization is mediated by second messenger-dependent protein kinases and GPCR kinases (GRKs). However, it remains to be determined whether mGluRs phosphorylation by GRKs and beta-arrestin binding are absolutely required for desensitization. Group 1 mGluRs endocytosis is both agonist-dependent and -independent. Agonist-dependent mGluRs internalization is mediated by a beta-arrestin- and dynamin-dependent clathrin-coated vesicle dependent endocytic pathway. The activation of Group 1 mGluRs also results in oscillatory Gq protein-coupling leading to the cyclical activation of phospholipase Cbeta thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and Ca(2+) release from intracellular stores. These glutamate receptor-stimulated Ca(2+) oscillations are translated into the synchronous activation of protein kinase C (PKC), which has led to the hypothesis that oscillatory mGluRs signaling involves the repetitive phosphorylation of mGluRs by PKC. However, recent experimental evidence suggests that oscillatory signaling is an intrinsic glutamate receptor property that is independent of feedback receptor phosphorylation by PKC. The challenge in the future will be to determine the structural determinants underlying mGluRs-mediated spatial-temporal signaling as well as to understand how complex signaling patterns can be interpreted by cells in both the developing and adult nervous systems.     

The presence of interleukin-2 receptor alpha in the serum of colorectal cancer patients is unlikely to result only from T cell up-regulation

It is unclear whether the presence of interleukin-2 soluble receptor alpha (IL-2 sRalpha) in the serum of colorectal cancer patients is solely due to T cell activation. In this study, we therefore investigated whether T cell activation, indicated by the up-regulation of the CD25 and HLA-DR markers, or cell-mediated immunity (CMI) were associated with increased serum levels of IL-2 sRalpha in patients with advanced colorectal carcinoma. The levels of serum IL-2 sRalpha and the proportion of T cells expressing HLA-DR (DR(+) T cells) were measured as markers for chronic activation. CMI was assessed by delayed-type hypersensitivity reaction (DTH) to intradermal injections of recall antigens. Eighty-seven colorectal liver metastases (CLM) patients and 23 'cancer-free' control subjects were studied. DR(+) T cells were found to be more prevalent ( P<0.0001) in CLM patients (median: 21.1%) than in controls (median: 3.4%), but DR(+) T cell up-regulation was not correlated with serum IL-2 sRalpha levels. CMI positivity was significantly reduced ( P=0.002) in CLM patients compared with controls, and this reduction was significantly associated ( P=0.05) with an increase in the number of DR(+) T cells. Although survival was significantly shorter ( P=0.0003) in patients with increased serum IL-2 sRalpha levels than in subjects with normal levels, no association was found between survival and DR(+) T cell up-regulation. These findings were consistent with the hypothesis of an additional source of serum IL-2 sRalpha other than T cell up-regulation in CLM patients -- either from other immune cells, or from tumour products.     

VSV transmembrane domain (TMD) peptide promotes PEG-mediated fusion of liposomes in a conformationally sensitive fashion

Helical instability induced by gly residues in the transmembrane domain (TMD) of G protein, the fusion protein of vesicular stomatitis virus (VSV), was speculated to aid in the later steps of the fusion process, because G protein with ala's substituted for the two TMD gly's was inactive (Cleverley, D. Z., and Lenard, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3425-30). Here we examine the conformations of synthetic peptides corresponding to fusion-active (GGpep) and inactive (AApep; G's replaced by A's) TMDs by CD spectroscopy, and then their effects on the kinetics of poly (ethyleneglycol) (PEG)-mediated fusion of small unilamellar vesicles. GGpep and AApep both assumed history-dependent, non-interconvertible ordered structures. Both peptides were largely helical under all conditions if derived from trifluoroethanol solutions, and aggregated in a beta-sheet form if derived from acetonitrile solutions. In solvent, detergents or lipid bilayers, GGpep showed a greater range of secondary structural features than did AApep. The two peptides had large but different effects on PEG-mediated fusion. Both enhanced the rate but not the extent of lipid mixing. AApep significantly inhibited the extent of fusion pore formation while GGpep had no effect. The initial rate of fusion was enhanced 6-fold by GGpep and less than 2-fold by AApep. Addition of 5 mol % hexadecane overrode all peptide-induced effects. We suggest that both GGpep and hexadecane promote pore formation by stabilizing the nonlamellar structures in fusion intermediates or initial small pores. AApep, which had fewer nonhelical features in its CD spectrum than GGpep, actually inhibited fusion pore formation.